Bone Marrow-Derived Cell Accumulation in the Brain in a Murine Model of Alzheimer's Disease

83 Pages·2013·2.05 MB·English

Bone Marrow-Derived Cell Accumulation in the
Brain in a Murine Model of Alzheimer’s Disease
by
Christine Barr
B.Sc. (Kinesiology), Simon Fraser University, 2010
THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in the
Department of Biomedical Physiology and Kinesiology
Faculty of Science
Christine Barr2013
SIMON FRASER UNIVERSITY
Summer2013
All rights reserved.
However, in accordance with the Copyright Act of Canada, this work may
be reproduced, without authorization, under the conditions for
“Fair Dealing.”Therefore, limited reproduction of this work for the
purposes of private study, research, criticism, review and news reporting
is likely to be in accordance with the law, particularly if cited appropriately.
Approval
Name: Christine Barr
Degree: Master of Science
Title of Thesis: Bone Marrow-Derived Cell Accumulation in the Brain
in a Murine Model of Alzheimer’s Disease
Examining Committee:
Chair: Dr. Parveen Bawa
Dr. Charles Krieger
Associate Professor
Senior Supervisor
Dr. Jonathan Choy
AssistantProfessor
Supervisor
Dr. Michael Silverman
Associate Professor, BISC
Simon FraserUniversity
External Examiner
Date Defended/Approved: August 9, 2013
ii
Partial Copyright Licence
Ethics Statement

The author, whose name appears on the title page of this work, has obtained, for the
research described in this work, either:
a. human research ethics approval from the Simon Fraser University Office of
Research Ethics,
or
b. advance approval of the animal care protocol from the University Animal Care
Committee of Simon Fraser University;
or has conducted the research
c. as a co-investigator, collaborator or research assistant in a research project
approved in advance,
or
d. as a member of a course approved in advance for minimal risk human research,
by the Office of Research Ethics.
A copy of the approval letter has been filed at the Theses Office of the University Library
at the time of submission of this thesis or project.
The original application for approval and letter of approval are filed with the relevant
offices. Inquiries may be directed to those authorities.
Simon Fraser University Library
Burnaby, British Columbia, Canada
update Spring 2010
Abstract
One difficulty in treating some neurological disorders is that manypharmaceuticals
cannot cross the blood brain barrier to reach affected areas. Human and animal studies
have shown that bone marrow transplantation can result in the engraftment of donor-
derived cells in the central nervous system (CNS) under certain conditions.
Understanding these conditions will allow us to optimize recruitment of bone marrow-
derived cells (BMDCs) to the CNS and, in the future, use these cells as vehicles for gene
delivery. Using a triple transgenic mouse model of Alzheimer’s Disease (AD), I studied
accumulation of amyloid-β, a pathological characteristic of AD, and association of
BMDCs with amyloid-β. There were difficulties in maintaining chimerism after bone
marrow transplantation in these mice. Reconstitutionwas achievedby depleting natural
killer cell activity in the host, suggesting that hybrid resistance may be present.
Keywords: Alzheimer’s Disease; bone marrow transplant; triple transgenic mouse;
hybrid resistance; amyloid-β
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Acknowledgements
I would like to thank my supervisor, Dr. Charles Krieger for all of the guidance
throughout this entire process. I would also like to thank Dr. Jonathan Choy for the
advice along the way and my external examiner, Dr. Michael Silverman for taking the
time to read andcritically review my thesis.
Thanks to my lab mates for the support, friendship and sharing of their knowledge with
me, Coral Lewis, John Manning, Sapana Thakore and Kyle Peake.
Thanks to the staff of the Animal Care Facility for showing me the ropes and making
sure mymice were always well taken care of.
And finally, thank you to my family for their loving support and encouragement.
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Table of Contents
Approval..........................................................................................................................ii
Abstract.......................................................................................................................... iii
Acknowledgements........................................................................................................iv
Table of Contents............................................................................................................v
List of Tables................................................................................................................. vii
List of Figures................................................................................................................viii
List of Acronyms.............................................................................................................ix
Introduction...................................................................................................................1
Alzheimer’s Disease........................................................................................................1
Amyloid-beta..........................................................................................................2
Hyperphosphorylated Tau......................................................................................4
Synaptic loss and neurodegeneration.....................................................................5
Inflammation in AD.................................................................................................6
Mouse Models of AD..............................................................................................8
Microglia........................................................................................................................10
Origin....................................................................................................................10
Function...............................................................................................................10
Role in AD............................................................................................................11
BM Transplantation.......................................................................................................13
Preconditioning.....................................................................................................13
Hybrid Resistance................................................................................................13
Conditions for BMDC accumulation in brain..................................................................16
Blood Brain Barrier...............................................................................................16
Accumulation of BMDCs in the CNS.....................................................................18
BM Transplantation Following Irradiation..............................................................19
BM Transplantation without Lethal Irradiation.......................................................21
Use of non-irradiative myeloablation with Busulfan...............................................22
Aims ....................................................................................................................23
Methods.......................................................................................................................24
Mice ............................................................................................................24
Pretreatment and BM transplantation...........................................................24
Chimerism monitoring..................................................................................25
Tissue preparation.......................................................................................25
Immunohistochemistry.................................................................................25
Statistics......................................................................................................26
Results.........................................................................................................................27
Triple transgenic AD mice express intracellular Aβ, extracellular Aβ plaques
and hyperphosphorylated tau.......................................................................27
Microglia accumulate around Aβ plaques.............................................................33
Bone marrow-derived cells accumulate in brain of AD mouse following
irradiation.....................................................................................................35
Bone marrow-derived cells associate with extracellular Aβ plaques.....................37
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Busulfan alone is not sufficient to promote sustained chimerism in 129/B6
hybrid AD mice............................................................................................38
Busulfan and Cyclophosphamide together give initial but temporary
engraftment of GFP+ BM..............................................................................40
Depletion of NK cell activity in the host allows for sustained peripheral blood
chimerism....................................................................................................42
Discussion...................................................................................................................46
Use of the 3x Tg Model of AD...............................................................................46
Quantification of Aβ and BMDC Association.........................................................51
Busulfan versus Irradiation...................................................................................53
Hybrid Resistance................................................................................................57
BBB in AD............................................................................................................58
References...................................................................................................................61
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List of Tables
Table 1. MHC haplotype of 129 and C57BL/6 mice.....................................................58
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List of Figures
Figure 1. Proteolytic pathway of amyloid precursor protein.............................................2
Figure 2. Incomplete set of self MHC in hybrid resistance............................................16
Figure 3. Components of the Blood Brain Barrier.........................................................17
Figure 4. Amyloid β immunoreactivity with age.............................................................28
Figure 5. 6E10 immunoreactivity in cerebral cortex......................................................29
Figure 6. Congo Red staining in APP/PS1 and 3x Tg brain..........................................30
Figure 7. Similar staining pattern of 6E10 and 11A1 at low power magnification..........31
Figure 8. High power image of intracellular Aβ.............................................................32
Figure 9. Phosphorylated tau in neurons of the hippocampus......................................33
Figure 10. Microglia associated with Aβ........................................................................34
Figure 11. Microglia associated with Aβ plaque............................................................34
Figure 12. Bone marrow derived cell accumulation after irradiation..............................36
Figure 13. GFP+ cell engraftment in brain of irradiated and transplanted AD mice
at 1 year of age............................................................................................37
Figure 14. GFP cells associated with Aβ plaques.........................................................38
Figure 15. Chimerism after 100 mg/kg BU pretreatment...............................................39
Figure 16. Chimerism after 80 mg/kg BU pretreatment and 129/B6 BM transplant.......40
Figure 17. Average chimerism following BU and CY pretreatment in 3x Tg AD
and B6 mice.................................................................................................41
Figure 18. Average chimerism in blood and BM of AD and B6 mice following BU
and CY pretreatment...................................................................................42
Figure 19. Average chimerism following BU and NK cell activity depletion...................43
Figure 20. Chimerism in NK cell depleted mice vs. non-NK cell depleted mice.............45
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